Skeidsvoll J, Ueland P M
Department of Clinical Biology, University of Bergen, Norway.
Electrophoresis. 1996 Sep;17(9):1512-7. doi: 10.1002/elps.1150170917.
Analytical parameters known to be important for the separation of DNA by capillary electrophoresis, including gel polymer concentration, electrical field strength and temperature, were investigated and optimized for the analysis of RNA molecules from 100 to 2000 bases. Denaturation, essential to obtain uniform and identifiable peaks, was accomplished by heating the sample in 80% formamide prior to electrophoresis and the presence of 2-8 M urea in the electrophoresis buffer. Efficient separations were obtained over a wide range of electrical field strengths and temperatures using the gel polymer hydroxypropylmethylcellulose (HPMC) as separation matrix. Low HPMC concentrations (< 0.3%) were suited for the separation of high molecular mass RNA (> 1000 bases) whereas higher HPMC concentrations were required for optimal separation of low molecular mass RNA. An optimized system was applicable for the separation of the predominating RNA populations (small RNA of 60-300 bases (as a group of unseparated peaks), 18S and 28S rRNA) in total RNA from a human glioma cell line. This is the first systematic investigation of electrophoresis of higher molecular mass RNA in capillaries, and motivates further studies to transfer electrophoresis of RNA to the capillary format.
研究了已知对毛细管电泳分离DNA很重要的分析参数，包括凝胶聚合物浓度、电场强度和温度，并针对100至2000个碱基的RNA分子分析进行了优化。通过在电泳前将样品在80%甲酰胺中加热以及在电泳缓冲液中加入2-8M尿素来实现变性，这对于获得均匀且可识别的峰至关重要。使用凝胶聚合物羟丙基甲基纤维素（HPMC）作为分离基质，在很宽的电场强度和温度范围内都能实现高效分离。低HPMC浓度（<0.3%）适合分离高分子量RNA（>1000个碱基），而分离低分子量RNA则需要更高的HPMC浓度才能实现最佳分离效果。优化后的系统适用于分离人胶质瘤细胞系总RNA中占主导地位的RNA群体（60-300个碱基的小RNA（以一组未分离的峰形式存在）、18S和28S rRNA）。这是首次对毛细管中高分子量RNA电泳进行的系统研究，并推动了将RNA电泳转移到毛细管形式的进一步研究。
